出芽短梗霉细胞多形性及影响细胞分化因素探索

Exploration of cell polymorphisms and factors influencing cell differentiation of Aureobasidium pullulans

摘要:

【背景】出芽短梗霉(Aureobasidium pullulans)是在生活史中有酵母状细胞生长阶段，并合成黑色素的一种黑酵母(Black yeast)，具有典型的细胞多形性，可分化形成酵母状细胞(yeast-like cell，YL)、膨大细胞(swollen cell，SC)、厚垣孢子(chlamydospore，CH)、菌丝(hyphae，HY)、念珠状菌丝(monilioid hyphae，MH)、有隔膜膨大细胞(Septate swollen cell，SSC)、分生组织状结构(meristematic structure，MS)，其中膨大细胞既可以作为生长的细胞类型，也可分化为其他的细胞类型。出芽短梗霉的形态分化是可调控的，调控因子有pH、温度、营养条件等。【目的】探究不同的氧气浓度、温度、盐浓度、营养水平对出芽短梗霉细胞形态的影响。【方法】利用显微镜、美兰染色等技术观察不同条件对出芽短梗霉细胞形态的影响。【结果】在完全无氧的试管底部菌体不能生长；在高层半固体表层(高氧气浓度)，酵母状细胞(YL)在营养丰富的生长初期出芽繁殖，在养分匮乏的培养后期诱导酵母状细胞(YL)经过膨大细胞(SC)形成厚垣孢子(CH)并合成黑色素；在营养丰富的生长初期，半固体试管浅表层和中间层(微好氧)低浓度氧气诱导YL经过SC形成HY侵入性生长。养分差异对菌体细胞多形性分化影响显著，环境适宜养分丰富(Yeast extract peptone dextrose medium，YPD)，以YL生长，不需要分化成HY；环境适宜养分不丰富(Potato dextrose agar，PDA)，分化成SC或HY以适应或逃离环境；环境不适宜养分匮乏时(Malt extract agar，MEA)，SC或HY分化成CH或MH进入休眠阶段。10% NaCl胁迫降低菌体生长速度，抑制色素合成、HY和MH的形成，并且细胞主要以YL生长繁殖。在相同质量浓度(10%)的KCl或Na2SO4渗透胁迫条件下，细胞多形性表型均为YL发达，HY及MH被抑制，说明高渗胁迫阻止了酵母状细胞向菌丝和厚垣孢子的分化。温度实验中，SC比YL耐高温，MS比SC耐高温。【结论】营养状态对出芽短梗霉细胞分化影响最大。

Abstract:

[Background] Aureobasidium pullulans belongs to “black yeast” and can synthesize melanin. A. pullulans displays characteristic polymorphic cell types, such as yeast-like cell (YL), swollen cell (SC), chlamydospore (CH), hyphae (HY), monilioid hyphae (MH), septate swollen cell (SSC), meristematic structure (MS). The swollen cell can be further differentiated into chlamydospore, monilioid hyphae, septate swollen cell and meristematic structure. The differentiation of A. pullulans can be regulated by various factors such as pH, temperature and nutritional conditions. [Objective] To study the effects of different oxygen density, temperature, osmotic pressure, pH value and nutrition level on the morphology of A. pullulan cells. [Methods] The microscopic techniques and methylene blue staining were used to observe the effects of different conditions on cell morphology of A. pullulans. [Results] Growth of A. pullulan was not observed under completely anaerobic condition. Under high oxygen condition, the yeast-like cells budded at the early nutrient-rich stage. However, once the nutrient was gradually used up, the yeast-like cells started to form swollen cell and then to chlamydospore. Under low oxygen condition, the yeast-like cell grew into HY through SC at the beginning of incubation when nutrients are rich. The different nutrient concentration had significant effects on the polymorphic differentiation of A. pullulans. Yeast-like cells were the main types of cells in the yeast extract peptone dextrose medium (YPD) medium with high nutrition and optimal ambient conditions. The YL cell differentiated into SC or HY to adapt to or escape the environment in the potato dextrose agar (PDA) medium. The malt extract agar (MEA) medium was nutrient deficient, therefore the SC or HY differentiated into dormant cell CH or MH. The 10% NaCl depressed the growth rate of A. pullulans and inhibited the synthesis of melanin. The effect of 10% KCl or 10% Na2SO4 on the cell differentiation of YL was the same as 10% NaCl, which suggests osmotic stress prevented the differentiation of yeast-like cells into hypha or chlamydospore. The SC was more resistant to high temperature than YL, but less resistant than MS. [Conclusion] Nutrition levels are the key factor regulating cell morphogenesis of A. pullulans.